This application claims priority to application PCT/FR98/02380 filed Nov. 6, 1998 which claims priority to France 97 14191 filed Nov. 6, 1997.
The present invention relates to a method for detecting and identifying and/or quantifying an enzymatic activity such as deaminase activity in a culture medium for microorganisms, to the compounds and detection agents which are suitable for this method, to the method for preparing these compounds and detection agents, as well as to the culture media for implementing said method.
The detection and identification of microorganisms are very important in particular in medicine, in the agrifoods industry or for environmental control (water, etc.). Microorganisms may be sought for their pathogenicity, as contamination indicators, for monitoring manufacturing methods, and the like.
The techniques for detecting and identifying microorganisms are currently based on the search for characteristic nucleotide sequences, the search for antigens or antibodies, culturing in selective or nonselective medium, or alternatively the search for metabolic and in particular enzymatic activities (for example osidase, esterase, peptidase, oxidase, etc. activities).
Usually, the methods for detecting and identifying and/or quantifying microorganisms combine several of these techniques. Culturing is thus used to multiply and select the desired microorganisms. In order to simply their detection, it has been proposed to demonstrate biochemical activities by introducing molecules which produce a coloration or a fluorescence, directly into the culture medium. Such media are referred to as detection media. The biochemical activities can be demonstrated by various methods, such as:
physicochemical modification of the medium: change in pH in the presence of a colored or fluorescent indicator (methylumbelliferone, etc.),
change in the redox potential, revealed with the aid of a colored indicator (tetrazolium salts, etc.) or a fluorescent indicator (EPO-424 293),
hydrolysis of molecules which release a colored compound or a fluorescent compound (indoxyl, naphthol, coumarin, etc.),
reaction of a molecule which is produced by the microorganisms with a compound which is present in the medium and which results in a coloration (detection of indole, James et al., 1986).
It is known that gelled (or solid) media are particularly suitable for culturing and isolating microorganisms from a sample, as well as for detecting xe2x80x9ctargetxe2x80x9d microorganisms in a mixture of microorganism taxa.
A method for differentiating bacteria of the Proteus group from other enterobacteria is known (Sverre Dick Henriksen, State Institute for Public Health, Bacteriological Department, Oslo, Norway, Jun. 6 1950). This method uses an equal amount, in gelled medium, of D- and L-phenylalanine in the presence of iron salt. In comparison with the urease assay, the green colored reaction obtained, although positive for identifying the Proteus group, is presented as being an assay which is laborious to use.
The document by GIAMMANCO and PIGNATO (xe2x80x9cRapid identification of microorganisms from urinary tract infections by beta-glucuronidase, phenylalanine deaminase, cytochrome oxidase and indole tests on isolation mediaxe2x80x9d, JOURNAL OF MEDICAL MICROBIOLOGY, vol. 41, No. 6, December 1994, pages 389-392) is also known, which discloses, for detecting a deaminase activity of bacteria of the Proteeae group, the use of natural amino acids, such as tryptophan and phenylalanine, in combination with an iron salt (FeCl3).
The document by MANAFI and ROTTER (xe2x80x9cA new plate medium for rapid presumptive identification and differentiation of Enterobacteriaceaexe2x80x9d, INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 14, No. 2, November 1991, pages 127-134) is also known, which discloses, for detecting a deaminase activity of bacteria of the Proteeae group, the use of tryptophan alone with iron salt (ammoniacal iron citrate). This document mentions, for other markers of enzymatic activity (4-methylumbelliferone, nitrophenyl), that a diffusion of these markers is detrimental. The only solution for minimizing the effect thereof is to incubate the media for a shorter time.
The document by PELOUX and LEFORT (xe2x80x9cLysine deaminase of the Proteus-Providencia group by means of the Edwards and Fife lysine-iron medium. Practical value of this medium for differentiating enterobacteriaxe2x80x9d, FEUILL. BIOL., vol. 13, No. 68, 1972, pages 37-42) is also known, which compares the activity of three amino acids (tryptophan, phenylalanine and lysine), for detecting deaminase, in combination with an iron salt.
The document by SIVOLODSKII (xe2x80x9cModification of a method for the determination of tryptophan deaminase and phenylalanine deaminase content in bacteriaxe2x80x9d, LAB. DELO., No. 3, 1982, pages 166-168) is also known, which proposes adding enzymatic hydrolysates of proteins, consisting of mixtures of natural amino acids, of peptides and of other compounds, in order to detect tryptophan and phenylalanine deaminase activities.
Patent application WO-A-92/00068 is also known, which discloses substituted histidine compounds which act as antagonists of angiotensin II receptors. These compounds, according to the formula (I) and the examples, comprise, inter alia, the substitution of the amine or carboxyl group.
U.S. Pat. Nos. 5,643,743 and 5,411,867 are also known, which disclose tryptophan for detecting tryptophanase, this enzyme being different from the deaminases.
U.S. Pat. No. 4,603,108 is also known, which discloses D,L-beta(p-nitrophenyl)alanine as a substrate for detecting phenylalanine deaminase. The reading of the reaction is carried out either at 480 nm, without adding a revelator, for phenylalanine deaminase, or by assaying ammonia for leucine deaminase.
Finally, U.S. Pat. No. 5,541,802 is known, which discloses, for detecting deaminases, the amino acids phenylalanine and tryptophan, which make it possible to obtain an orangey color which diffuses in the medium.
Either these documents disclose the use of natural amino acids, which is incompatible with a limitation of the diffusion of alpha-keto acid. The only solution proposed by certain of these documents comes down to limiting the incubation time, which is very prejudicial to the quality of the detection. However, the limitation of diffusion is evidence of differentiation of several microbial colonies present in the same medium. Or these documents disclose the use of artificial and modified amino acids which do not satisfy formula (I) of the invention, and which can, in addition, provide a different application, such as for example antagonism with angiotensin II receptors.
Despite all the biochemical assays currently on the market, it turns out that currently there are no means available, which are particularly well-suited and easy to implement, in particular in gelled medium, for detecting and identifying and/or quantifying, in a multimicrobial culture, an enzymatic activity such as a deaminase activity of microorganisms.
The present invention thus intends to resolve this problem.
A first subject of the invention is a method for detecting and identifying and/or quantifying an enzymatic activity such as deaminase activity of a microorganism, according to which an inoculum which is capable of containing a microorganism with a deaminase activity is brought into contact with a culture medium for microorganisms, the culture medium comprising at least one detection agent for demonstrating, by forming a colored product with a revealing agent, an enzymatic activity such as deaminase activity; said detection agent is a cyclic L-amino acid of following general formula (I): 
in which:
R represents a cyclic amino acid radical, substituted with 1 to 3 groups X, which are identical or different,
X represents a group which limits the diffusion of the xcex1-keto acid produced by the deamination of the cyclic amino acid,
the compound of formula (I) being able to be substituted with various groups which do not interfere with the function of the group X.
xe2x80x9cGroup which limits the diffusionxe2x80x9d is intended to mean:
any group of hydrophobic type which limits the diffusion in a hydrophilic medium, or
any group which makes it possible to associate with, via weak bonds, in particular hydrophobic bonds, or to bind to, via one or more covalent bonds, in particular thiol bonds, constituents of the cells of the microorganisms, such as the wall, the membrane, the proteins, etc.
As an assay defining the groups which limit the diffusion, a cyclic L-amino acid and a corresponding detection agent according to the invention are respectively inoculated, as a spot, at the center of two Petri dishes. The diameters of the diffusion of the xcex1-keto acids produced by the enzymatic activity such as the deaminase activity of the inoculant are measured. The diameter of diffusion of the detection agent according to the invention should be less than that of the cyclic L-amino acid; it will then be considered that the detection agent limits the diffusion of the a-keto acid produced by the deamination of the L-amino acid.
xe2x80x9cCyclic amino acid radical Rxe2x80x9d is intended to mean cyclic or heterocyclic radicals R such as indole, phenyl, hydroxyphenyl and imidazole, which can undergo a reaction of substitution with the group X. These amino acids include amino acids which are natural or synthetic, or modified, in particular by substitution.
xe2x80x9cGroup which does not interfere with the function of the group Xxe2x80x9d is intended to mean any group which does not prevent the group X from limiting the diffusion of the xcex1-keto-acid.
In addition, as used herein, the term xe2x80x9csubstituentxe2x80x9d does not include hydrogen.
In the method according to the invention, adding at least one detection agent in suitable concentrations does not inhibit the multiplication of microorganisms in the appropriate culture media. The detection agent can thus be used by adding it to the culture medium of the microorganisms before the start of the culture or at the start of culture.
One of the important advantages of the method using detection agents according to the invention is that in the presence of an enzymatic activity such as deaminase activity, it gives, after adding a revealing agent, colored products which do not diffuse in the medium, in particular in a gelled medium.
The method according to the invention can thus advantageously be used in gelled medium. It can also, of course, be used in liquid medium or on a solid support in the absence of gelling agent.
The method according to the invention makes it possible, in a multimicrobial medium, to detect and identify, or even quantify microorganisms with an enzymatic activity such as deaminase activity, and to do so without interfering with the detection of other microorganisms. On the contrary, the method according to the invention makes it possible, with the set of enzymatic reactions produced, to reveal distinctly, with colors which define the type of microorganism, the microorganism(s) present in the medium.
The method according to the invention thus provides the advantage, besides that of detecting and identifying an enzymatic activity such as deaminase activity in a culture medium, of allowing a simple and inexpensive implementation.
In a preferential embodiment according to the invention, the revealing agent is a cation salt.
The cation salt, which makes it possible to reveal the detection agent by forming a colored product, can be added to the culture medium at the same time as the detection agent, or indeed after culturing the microorganisms. In fact, persons skilled in the art will know how to determine, according to the culture medium used, whether it is more advantageous, or whether it is preferable, to add the cation salt at the same time as the detection agent or after culturing the microorganisms. However, adding the revealing agent to the culture medium at the same time as the detection agent is preferred, in particular in gelled medium.
The reaction which takes place between the detection agent and the deaminase, and then the revealing agent, in the presence of flavoprotein, when for example phenylalanine is used as a detection agent is the following general reaction: 
The phenylpyruvic acid obtained, which is an alpha-keto acid, combines with iron chloride, which acts as a chelating agent, to give a green coloration. Thus, when a tube containing such a composition is exposed to atmospheric oxygen, the coloration density increases.
According to the nature of the amino acid used, the final color obtained varies. For example, in the case of histidine, the coloration is brown.
This reaction is respected when the phenyl group is replaced with another ring and is substituted one to three times with a group X according to the formula (I).
The microorganisms which are detected and identified and/or quantified by enzymatic activity such as deaminase activity according to the method of the invention belong, in particular, to the group Proteus.
In a very preferential embodiment according to the invention, at least one other detection agent for demonstrating, by forming a colored or fluorescent product, an enzymatic activity which is different from that demonstrated by the compound of general formula (I), is also added to said culture medium. It can be for example an esterase, oxidase or peptidase activity. Additional information can also be obtained, in connection with an absence of coloration (or of fluorescence) or in connection with a coloration which is modified with respect to the coloration obtained with a single enzymatic substrate. The other detection agent chosen will have properties which are different from those of the detection agents of formula (I) according to the invention. For example, another detection agent which is capable of giving a reaction product with a color which is different from the color obtained with the detection agents of formula (I) will be chosen. The other detection agent (or second detection agent) will thus make it possible to reveal, due to its own color or due to its fluorescence, the presence of an enzymatic activity for which it is specific. If the enzymatic activity such as deaminase activity, which is detectable by the detection agent [sic] of formula (I) according to the invention, is also present, and can be revealed with a characteristic color, a modified coloration, which is different from said characteristic color and which is also different from said own color given by the second detection agent, will be obtained. Examples of using several detection agents in the same culture medium are given in the following examples. Obviously, the results can vary with the choice of the detection agent according to the invention which is used, the second (or more) detection agent used and the various microorganisms present in the culture medium.
The other detection agents which are used to demonstrate different enzymatic activities are in particular, but not exclusively, indoxyl, coumarin, resorufin, naphthol, naphthylamine, nitrophenol, nitroaniline, rhodamine, hydroxyquinoline, fluorescein, etc. derivatives.
Among these other detection agents which can be used in combination with the detection agents according to the invention, mention may be made in particular of 5-bromo-4-chloro-3-indolyl-xcex2-D-glucuronide, 6-chloro-3-indolyl-xcex2-D-glucoside, L-alanine-7-amino-4-methyl-coumarin, 4-methylumbelliferyl-N-acetyl-xcex2-D-galactos-aminide, resorufin-xcex2-D-galactoside, xcex2-naphthyl sulfate, naphthol AS-BI xcex2-D-galactoside, L-alanine xcex2-naphthyl-amide, O-nitrophenol-xcex2-D-galactoside, carboxybenzoyl-L-arginine-p-nitroanilide, rhodamine 110 bis-(L-leucine amide) and fluorescein diacetate.
A second subject of the invention are the compounds having the following general formula (I): 
in which:
R represents a cyclic amino acid radical, substituted with 1 to 3 groups X, which are identical or different,
X represents a group which limits the diffusion of the xcex1-keto acid produced by the deamination of the cyclic amino acid,
the compound of formula (I) being able to be substituted with various groups which do not interfere with the function of the group X, with the exception of the compounds N-im-benzyl-L-histidine, 1- and 3-methyl-L-histidine, O-benzyl-L-tyrosine, O-carboxybenzoyl-L-tyrosine, O-dansyl-L-tyrosine, O-methyl-L-tyrosine and 1-, 4-, 5-, 6- and 7-methyl-L-tryptophan.
The abbreviations xe2x80x9cN-imxe2x80x9d for an L-histidine compound, and xe2x80x9cN-imxe2x80x9d for an L-tryptophan compound signify that the nitrogen atom of the imidazole nucleus (His) or the nitrogen atom of the indole nucleus (Trp) is substituted.
A third subject according to the invention is a detection agent comprising at least one compound of following general formula (I): 
in which
R represents a cyclic amino acid radical, substituted with 1 to 3 groups X, which are identical or different,
X represents a group which limits the diffusion of the xcex1-keto acid produced by the deamination of the cyclic amino acid, the compound of formula (I) being able to be substituted with various groups which do not interfere with the function of the group X.
In a preferential embodiment, the detection agent comprises at least one compound of following general formula (I): 
in which R is substituted with a group X, and X is chosen from hydrophobic groups.
In an even more preferred embodiment, the detection agent comprises at least one compound of following general formula (I): 
in which R is substituted with a group X, and X is chosen from methyl, benzyl, carboxybenzoyl, dansyl, naphthalene-sulfonyl, toluene-sulfonyl and mesitylene-sulfonyl.
A fourth subject according to the invention is the method for preparing compounds and detection agents according to the invention of general formula (I) and as respectively defined above, comprising the following steps:
(a)xe2x80x94formylation of the residue R,
(b)xe2x80x94addition of a salt of X onto the residue R formylated according to (a),
(c)xe2x80x94deformylation of the residue R substituted according to (b).
A fifth subject according to the invention is a culture medium for microorganisms comprising, besides the ingredients required for culturing said microorganisms, at least one detection agent as defined above.
The detection agents of cyclic X-L-amino acid formula are used at weight concentrations which are sufficient to give observable colored reactions. These concentrations can be determined by routine experiments by persons skilled in the art.
In a preferential embodiment, the weight concentration of the detection agent(s) in the culture medium is between 0.025 and 5 g/l of culture medium.
In a very preferential embodiment, the weight concentration of the detection agent(s) in the culture medium is between 0.1 and 2 g/l, preferably between 0.3 and 0.6 g/l.
In another preferential embodiment, the culture medium also comprises a revealing agent, preferably a cation salt, for example ammoniacal iron citrate. The concentration of the revealing agent is not limiting; it can be lower than, equal to, or higher than that of the detection agent of formula (I).
In another preferential embodiment according to the invention, the culture medium is in a gelled form.
In a very preferential embodiment according to the invention, the culture medium also comprises at least one other detection agent for demonstrating, by forming a colored or fluorescent product, an enzymatic activity which is different from that demonstrated by the compound of general formula (I).
As an example of a gelled medium, O-toluene-sulfonyl-L-tyrosine at 0.5 g/l and iron citrate at 0.5 g/l can be incorporated into the culture medium, in the following medium:
this medium being able to be inoculated with various Gram-negative or Gram-positive microorganisms. Only the strains which belong to the group Proteus form brown colonies after 18 to 24 hours of incubation. The strains which do not belong to the group Proteus form colonies which conserve the appearance that they have on the same medium lacking in O-toluene-sulfonyl-L-tyrosine.